Factor VIII (fVIII) is a serum protein that acts as a critical cofactor and regulator of the intrinsic blood coagulation pathway. Factor VIII acts by nucleating the assembly of a membrane-bound proteolytic complex that contains the factor IXa protease on the surface of activated platelets. This complex activates factor X as part of a proteolytic cascade that generates fibrin polymers. Prior to its own activation, factor VIII circulates in a tight, stable complex with von Willebrand Factor. A wide variety of inheritable coagulation deficiencies are associated with mutations in factor VIII (hemophilia A), factor IX (hemophilia B) and von Willebrand Factor (von Willebrand Disease). Recent studies have reported the high resolution structure of the C2 membrane-binding domain of factor VIII, the structural characterization of hemophilia-associated missense mutations found in the fVIII C domains, the structure of a complex between fVIII and a hemophilia patient-derived antibody inhibitor, and a detailed analysis of the structural genomic relationships throughout the discoidin fold superfamily (of which the fV and fVIII C domains are members). The first specific aim for this project renewal is to characterize the effects of hemophilia-associated point mutations throughout the solvent-exposed surface of the factor VIII C2 domain on its stability, its membrane binding affinity and its association with von Willebrand Factor. These experiments will test the hypothesis that a specific subset of these mutations cause deficiencies in factor VIII membrane binding or vWF association. The second specific aim for this project is to determine the three-dimensional structure of full-length fVIII heterodimer. This structure will be used to study the role of domain motions involved in membrane binding by fVIII, to perform detailed analyses of the structural epidemiology of hemophilia-associated point mutations across the entire structure of the fVIII heterodimer, and to provide a high quality structural model for future crystallographic studies of protein complexes with factor VIII. The third specific aim for this project is to determine the structure of the N-terminal, D'-D3 domain from von Willebrand Factor, alone and/or in complex with factor VIII heterodimer.